The objective of this proposal is to understand the regulation of keratinization using filaggrin and keratins as marker proteins of differentiation of oral epithelia and skin. These epithelia exhibit tissue-specific differentiation which is altered in mucosal and epidermal diseases. Keratohyalin granules are organelles present only in keratinizing epithelia (palate, gingiva, and epidermis). They contain profilaggrin, a precursor of filaggrin, that causes keratin filament aggregation and dense packing into cells of the stratum corneum. The presence of filaggrin seems to correlate with differentiation-specific keratins and both exhibit regional variation in oral tissues.
Specific aims are (1) to test the correlation of profilaggrin and keratin expression in several situations amenable to study: (a) in vivo in rat epidermis and oral epithelia, (b) in vitro in a cultured rat epidermal cell line and in cultured oral epithelial cells, and (c) in a mutant mouse (repeated epilation, Er) with altered keratinization and oral defects, and (2) to investigate control mechanisms for expression of profilaggrin mRNA and protein, and post-translational conversion to filaggrin. Control mechanisms will be studied in vitro in cultured oral and epidermal cells utilizing inhibitors of ion flux, agents that interact with protein kinase C, and alteration of Vitamin A levels. The overall hypothesis is that keratinization is regulated by cell surface events, modulated by these agents; that these events are critical for control of transcription, translation, and enzymatic processing of profilaggrin; and that the expression of tissue-specific keratins and filaggrin have some regulatory events or processes in common. Profilaggrin genomic clones will be selected and characterized for regulatory sequences. Detailed studies of profilaggrin mRNA expression will reveal transcriptional and post-transcriptional regulatory mechanisms. Methods include the use of cDNA clones for gene selection and DNA sequencing; hybridization studies and in situ hybridization to determine the quantity and localization of mRNA; labeling studies, SDS-PAGE, and immunological methods to investigate profilaggrin, filaggrin, and other proteins; and assays for kinases, phosphatases, and proteases involved in post-translational modification. Completion of these studies will result in a significant advance in the understanding of the cell biology and keratinization of normal oral epithelia and skin.
Showing the most recent 10 out of 30 publications
